Pool-and-Split Vessel and Synthesizer for the Synthesis of Barcoded Beads used in Single-Cell Applications Abstract/Summary As the single-cell genomics?and in particular RNA-sequencing (scRNA-Seq)?field has matured, methods to work in high throughput have been developed. Many of these critically rely on solid support beads to capture cellular RNA targets in microwells or reverse emulsions. An essential requirement on these beads is a DNA barcode that is approximately unique to each bead and ties the contents of that bead back to a single original cell. The DNA barcode synthesis requirements are not compatible with state-of-the-art DNA synthesizers, and as such, barcode truncation and lack of complexity abound, impacting method fidelity. Current synthesized barcoded primer beads have quality issues that can be traced, in part, to the manual handling of the beads during barcode generation, or ?pool-and-split? steps, and to the incorporation inefficiency of nucleotides during the chemical synthesis of the growing oligonucleotide chains. We propose to develop an automated pool-and-split system to synthesize uniquely barcoded primer beads with a high content of full-length sequences by eliminating all manual handling and increasing overall bead coupling efficiency and recovery yields. Abstract Project Summary.docx Page 1 of 1